Inducer of apoptosis

ABSTRACT

This invention relates to the use of a mutant E2 polypeptide to induce apoptosis (a form of programmed cell death) in cells. The mutant E2 derived polypeptide is p53 binding deficient. Preferably it is unable to bind p53; or able to bind p53 but unable to induce p53-dependent apoptosis.

FIELD OF THE INVENTION

This invention relates to a method of inducing apoptosis (a form ofprogrammed cell death), particularly in cells that containpapillomavirus DNA, using a mutated papillomavirus E2 protein; tomethods of killing cells using such mutant proteins and to E2 derivedpolypeptides.

BACKGROUND

Papillomaviruses (PV) are DNA viruses that have a double strandedcircular genome containing several open reading frames (ORFs) whichencode products including the E6, E7, and E2 proteins (Meyers, G., etal. (1995) Human papillomaviruses, 1995 compendium. Los Alamos NationalLaboratory, Los Alamos, N. Mex., USA). These viruses infect epithelialcells and induce the formation of hyperproliferative lesions in whichthe viral DNA is usually present as an episome “i.e. autonomous selfreplicating DNA” (Lorincz A. T. et al. (1992) Obstet. GynecoL 79,328-337). At least 95 different types of human PV (HPV) have beenidentified, many of which infect the genital tract and produce genitalwarts (Van Ranst M., et al (1992) J. Gen. Virol. 73, 2653-2660). OtherHPV types are associated with cancer. For instance, HPV DNA can bedetected in virtually all cervical cancers (99.7%) and these viruses aregenerally acknowledged to be the causative agent of this disease(Walboomers J. M., et al. (1999) J. Pathol. 189, 12-19). HPVs are alsothought to be involved in a variety of other diseases including: cancerof the vulva, oral cancer, skin cancer, and cancer of the esophagus(Basta A., et al. (1999) Eur. J. Gynaecol Oncol. 20, 111-114: Miller C.S., & Johnstone B. M. (2001) Oral Surg. Oral Med Oral Pathol. OralRadiol. Endod. 91, 622-635: Biliris K. A., et al. (2000) Cancer Lett.161, 83-88: Layergne D., & de Villiers E. M. (1999) Int. J. Cancer 80,681-684).

In contrast to the episomal HPV DNA present in HPV-infected cells, theHPV DNA present in HPV-transformed cancer cells is often integrated intothe host genome (Dürst M., et al. (1985) J. Gen. Virol. 66, 1515-1522:Kalantari M., et al. (2001) Diagn. Mol. Pathol. 10, 46-54). However,cervical cancer cells continue to express the HPV E6 and E7 ORFs and theproducts of these oncogenes act to increase cell proliferation andpromote cell immortalization (Hawley-Nelson, P., et al. (1989) EMBO J.8, 3905-3910: Scheffner, M., et al. (1990) Cell 63, 1129-1136: Dyson,N., et al. (1989) Science 243, 934-936), essential initial steps intumourigenesis.

The papillomavirus E2 ORF encodes a sequence-specific DNA bindingprotein that regulates viral gene expression and which is also requiredfor efficient viral DNA replication (Bouvard V., et al. (1994) EMBO J.13, 5451-5459: Frattini M. G. & Laimins L. A. (1994) Proc. Natl. Acad.Sci. USA 91, 12398-12402: Berg M. & Stenlund A. (1997) J. Virol. 71,3853-3863). The E2 protein regulates transcription of the E6 and E7oncogenes and can thereby affect cell proliferation (Dowhanick J. J., etal. (1995) J. Virol. 69, 7791-7799: Sanchez-Perez A. M., et al. (1997)J. Gen. Virol. 78, 3009-3018: Desaintes C., et al. (1999) Oncogene 18,4538-4545). The integration of HPV DNA into the host genome oftendisrupts the E2 ORF and/or blocks the correct expression of E2 (DürstM., et al. (1985) J. Gen. Virol. 66, 1515-1522: Rose B. R., et al.(1997) Gynecol. Oncol. 66, 282-289). This is thought to lead toover-expression of E6 and E7, which in turn leads to tumourigenesis.Consistent with this view, over-expression of E2 proteins in cervicalcancer cells can repress E6 and E7 expression, resulting in apoptoticcell death and growth suppression (Dowhanick J. J., et al. (1995) J.Virol. 69, 7791-7799: Francis D. A., et al. (2000) J. Virol. 74,2679-2686: Nishimura A., et al. (2000) J. Virol. 74, 3752-3760). Thisfinding gave rise to the proposal that E2 could be useful in thetreatment of cervical cancer and other HPV-induced diseases. However,the E2 protein can also induce apoptosis in cells that do not containHPV DNA (Webster K., et al. (2000) J. Biol. Chem. 275, 87-94).

p53 is a cellular tumour suppressor protein that is inactivated bymutation in around half of all human tumours (reviewed by Cox L. S. &Lane D. P. (1995) Bioessays 17, 501-508). In response to a number ofstimuli including ionising radiation, cell stress, or viral infection,the p53 protein can mediate either cell cycle arrest or apoptosis. Cellsfrom HPV-induced tumours usually contain wild-type p53 and can respondto signals that induce p53 activity (Butz K., et al. (1995) Oncogene 10,927-936: Webster K., et al. (2000) J. Biol. Chem. 275, 87-94). The E2protein from at least one HPV type binds to p53 (Massimi P., et al.(1999) Oncogene 18, 7748-7754) and this E2 protein can also inducep53-dependent apoptosis (Webster K., et al. (2000) J. Biol. Chem. 275,87-94). Here we show that a mutant of the E2 protein that binds weaklyto p53 is capable of inducing apoptosis in HPV-transformed cells but isincapable of inducing apoptosis in non-HPV transformed cells. This novelmutant of E2 could be useful in cancer therapy, in the treatment ofprecancerous lesions, and in the treatment of HPV infections. Inparticular the invention provides a treatment for HPV infection,particularly the pre-cancerous condition known as cervicalintraepithelial neoplasia. Currently, many women diagnosed with thiscondition are not immediately treated but are regularly monitored forprogessing infection. Early treatment using the methods of thisinvention would alleviate much stress as well as reducing thepossibility of deterioration to a more serious form of the pre-cancerouscondition which has a higher risk of developing into cancer of thecervix. In contrast to such active treatments the methods andcompositions can be employed prophylactically against cervical cancer.

WO00/02693 (The University of Bristol) discloses methods andcompositions for inducing cell death in HPV-transformed andnon-HPV-transformed cells using E2 polypeptides. There is no disclosureof methods or mutations that could target E2-induced apoptosis toHPV-transformed cells or HPV-infected cells.

WO98/01148 (Harvard) discloses methods and compositions for interferingwith the proliferation of cells infected with and/or transformed by PV.There is no disclosure of the p53 status of the cells, the induction ofapoptosis in the treated cells, or the effects of E2 on HPV-negativecells.

WO94/04686 (Biogen) describes a method for the delivery of proteins,including HPV E2 polypeptides, to cells based on the HIV TAT protein.There is no disclosure of the p53 status of the cells, the induction ofapoptosis in the treated cells, or the effects of E2 on HPV-negativecells.

WO92/12728 (Biogen) discloses non-functional E2-derived polypeptides,specifically E2 trans-activation repressors, which form heterodimerswith normal E2 and block its function in HPV-infected cells. There is nodisclosure of the p53 status of the cells, the induction of apoptosis inthe treated cells, or the effects of E2 on HPV-negative cells.

DISCLOSURE OF THE INVENTION

According to one aspect of the invention there is provided a method ofkilling cells that contain HPV DNA, comprising contacting the cells witha p53 binding-defective PV E2-derived polypeptide. Such a polypeptidebinds to p53 with less affinity (if at all) than a wild-type or nativeHPV 16 E2 protein. The cells may be PV-transformed or PV-infected. Theinvention applies to all HPV types.

According to another aspect of the invention there is provided a methodof inducing apoptosis in PV-transformed cells and/or PV-infected cellscomprising contacting the cells with a p53-binding-defective PVE2-derived polypeptide.

According to another aspect of the invention there is provided a methodof killing PV-transformed cells and/or PV-infected cells comprisingcontacting the cells with a DNA sequence encoding ap53-binding-defective PV E2-derived polypeptide.

According to another aspect of the invention there is provided a methodof killing PV-transformed cells and PV-infected cells comprisingcontacting the cells with a p53 binding-defective PV E2-derived proteinthat is unable to bind p53 or a functional portion thereof.

According to a further aspect of the invention there is provided amethod of killing PV-transformed cells and PV-infected cells comprisingcontacting the cells with a nucleotide sequence encoding a p53-bindingdefective PV E2-derived polypeptide fused to VP22 or a derivativethereof. VP22 is a Herpes Simplex Virus-1 protein that can be used toefficiently deliver other polypeptides to mammalian cells (Elliot, G andO'Hare, P. (1997) Cell, 88: 223-233 and WO00/53722). It is advantageousbecause it should allow the delivery of the E2-derived protein to alarge number of cells compared to conventional gene therapy techniques.Suitable derivatives of or alternatives to VP22 having a similartransport function may be determined by the skilled worker. Othernon-viral methods of delivering E2 derived polypeptides includepenetratin or liposomes. Viral methods of delivering E2 derivedpolypeptides in adenovirus, adeno-associated virus, retrovirus, and poxvirus. Preferably, the nucleotide sequence encodes a p53-bindingdefective PV E2-derived polypeptide fused to VP22. More preferably, thep53-binding defective E2 derived polypeptide is delivered as ahistidine-tagged VP22 fusion protein. Most preferably the nucleotidesequence encodes a PV E2-derived polypeptide, that is unable to bindp53, fused to histidine-tagged VP22.

According to another aspect of the invention there is provided the useof a p53-binding defective PV E2-derived polypeptide in a pharmaceuticalcomposition for the active or prophylatic treatment or amelioration ofcervical cancer. The pharmaceutical composition would comprise suitablediluent or carrier. It is preferred that the composition is in the formof a cream or spray which can be applied directly to an infected areaespecially the cervical area in a female human subject.

According to another aspect of the invention there is provided the useof a p53 binding defective E2-derived polypeptide in a pharmaceuticalcomposition for the active or prophylactic treatment or amelioration ofgenital warts.

According to another aspect of the invention is provided the use of aDNA sequence encoding a p53-binding defective E2-derived polypeptide ina pharmaceutical composition for the active or prophylactic treatment oramelioration of cervical cancer.

According to another aspect of the invention there is provided the useof a DNA sequence encoding a p53-binding defective E2-derivedpolypeptide in a pharmaceutical composition for the active orprophylactic treatment or amelioration of genital warts.

According to another aspect of the invention there is provided the useof a p53 binding defective E2-derived polypeptide in a pharmaceuticalcomposition for the treatment or amelioration of precancerous lesions.

According to another aspect of the invention is provided the use of aDNA sequence encoding a p53-binding defective E2-derived polypeptide ina pharmaceutical composition for the treatment or amelioration ofprecancerous lesions.

Preferably a substantial portion, for example greater than 50%,preferably more than 80%, most preferably more than 90%, of the cellsare killed in a method of inducing apoptosis or killing cells accordingto the invention.

Preferably the cells in which apoptosis is induced are tumourigenic.Where the cells are cancerous, the cancer may be selected from cervicalcancer, cancer of the vulva, oral cancer, cancer of the oesophagus.

The HPV DNA may be integrated into the cell genome or may be present inthe cell as episomes. Alternatively, the HPV DNA may be present in thecell as both episomes and integrated DNA.

The cells may be mammalian. The cells may be cervical, vulval orepidermal. Preferably, the cells are human cells. More preferably thecells are in a mammalian subject, most preferably a human subject.

In methods and uses in accordance with the invention, the p53-bindingdefective E2-derived polypeptide may be able to bind p53 but unable toinduce p53-dependent apoptosis.

According to another aspect of the invention there is provided ap53-binding defective E2-derived polypeptide for use in methods and usesof the invention in which the polypeptide is mutated at at least one ofpositions Asp338, E340 (Glu 340), Trp341, and Asp344 of the native HPV16 protein amino acid sequence.⁹ Preferably the p53-binding defectiveE2-derived polypeptide is unable to bind p53. Where the polypeptide isable to bind p53, it may be unable to induce p53-dependent apoptosis.The PV from which the p53-binding defective polypeptide is derived maybe HPV type 16, 18, 33, 31, 6, 11, 2, 4, 1, or 7. Preferably, the PV isan animal, most preferably a mammalian PV, especially human PV. Thepolypeptide may have the same or similar length as a native E2 proteinor may be substantially shorter or longer. More particularly, where thepolypeptide is shorter than a native E2 protein the N terminal portionof the E2 protein may be truncated by 10 to 20 amino acids whereas inthe C terminal portion the truncation may be 20 to 40 amino acids. Wherethe polypeptide is longer than a native E2 protein it may be 10 or moreamino acids longer than at the N terminal end—for example because itincludes a histidine tag or is attached to VP22.

Alternatively the native sequence may be altered at one or any otherpositions that are important for the E2-p53 interaction. Mutations andalterations may be in the form of insertions, deletions orsubstitutions. Amino acids in a native sequence may be modified toenhance useful properties such as inhibition of p53 binding, stability,immunogenicity, expression. Polypeptides in accordance with theinvention may be produced by recombinant organisms or chemicallysynthesized.

A preferred polypeptide in accordance with the invention has the aminoacid sequence shown in FIG. 2A, 2B, 2C, or 2D.

The invention also provides nucleotide sequences encoding p53-bindingdefective E2-derived polypeptides of the invention. A preferrednucleotide has the nucleotide sequence shown in FIG. 2A, 2B, 2C, or 2D.

The nucleotide may encode an HPV 16 E2 derived polypeptide mutated atpositions Asp338, Glu 340, Trp341, and Asp344 of the native sequence orany other positions that are important for the E2-p53 interaction.

The p53 binding-defective PV E2-derived polypeptide may be delivered asa VP22 fusion protein. Other delivery methods such as gene therapy andantibody delivery are contemplated. For example, adenovirus,adeno-associated virus, or retroviruses may be used. The skilled workerwill be able to select suitable methods.

Definitions

In this specification, the following expressions are used with thefollowing non-limiting meanings given by way of explanation:

-   1. “PV positive” a cell that has been infected or transformed by a    PV. “PV negative” has an opposite meaning.-   2. “Protein” and “polypeptide” are used interchangeably although    protein may be considered to be a naturally occurring polypeptide.-   3. “HPV DNA” means a DNA from an HPV comprising a complete open    reading frame (ORF) or a significant amount, such as about 100 bp,    of non-coding sequence from a naturally occurring HPV genome.-   4. “Apoptosis” is a form of cell death chiefly though not    exclusively characterised by plasma membrane blebbing, chromatin    condensation, and the formation of apoptotic bodies (cell bodies    with sub-GO DNA content).-   5. “PV infected” cells are non-malignant (non tumourigenic) cells    containing HPV DNA.-   6. “PV transformed” cells are malignant cells that contain HPV DNA.

A method of inducing cell death and products in accordance with theinvention will now be described, by way of example only, with referenceto the accompanying drawings FIGS. 1 to 8 in which:

FIG. 1 shows the DNA for and amino acid sequence of a p53binding-defective E2-derived polypeptide HPV 16 E2p53mCt. The mutatedbases and amino acids are shown in bold and are underlined;

FIG. 2 shows the DNA for and amino acid sequence of HPV 16 E2p53m andother E2 derived polypeptides in accordance with the invention. Themutated bases and amino acids are shown in bold and are underlined;

FIG. 3 shows the HPV 16 E2 DNA binding domain and the positions at whichamino acids were mutated to create E2pS3m and E2p53mCt;

FIG. 4 shows the binding of labelled p53 to the E2Ct protein and theE2p53mCt protein;

FIG. 5 shows the effects of the E2 and E2p53 proteins on a variety ofHPV-transformed and non-HPV transformed cell lines;

FIG. 6 shows the ORF and amino acid sequence of HPV16 E2;

FIG. 7 shows the effects of VP22 and VP22-E2 fusion proteins onHPV-transformed cells; and

FIG. 8 shows the effects of a VP22-E2 fusion protein produced inE2-insensitive COS-7 cells on HPV-transformed cells.

1. EXPERIMENTAL PROCEDURES—GENERAL

a. Plasmids

The plasmids pWEB-E2 and pCMX-GFP3 express the HPV 16 E2 protein and thegreen fluorescent protein, respectively (Webster K., et al. (2000) J.Biol. Chem. 275, 87-94). The p53 expression vector pCB6-p53 was suppliedby Dr K. Vousden (NCI-FRDC, USA). The plasmid pBluescript-p53 containsthe wild-type p53 coding sequence downstream of the T7 promoter.pBluescript-p53 was created by cloning a BamHI fragment carrying the p53cDNA from pC53-SN3 (supplied by Dr B. Vogelstein (Johns Hopkins OncologyCenter, USA)) into the unique BamHI site in pBluescript II (Stratagene).The plasmid pKK-E2Ct expresses the DNA binding domain of the HPV 16 E2protein (amino acids 280 to 365) in bacterial cells (Webster K., et al.(2000) J. Biol. Chem. 275, 87-94). The plasmid pKK-E2p53mCt was createdby replacing the E2 sequences between the unique PstI and HindIII sitesin pKK-E2Ct with three double stranded synthetic oligonucleotides withcomplementary ends. The synthetic oligonucleotides introduced threeamino acid changes into the E2 sequence: Asp338 to alanine, Trp341 toalanine, and Asp344 to alanine (FIG. 1). Further changes that arecontemplated include Glu340 to alanine, Gln342 to alanine, Gln345 toalanine, and Arg343 to alanine. The plasmid pWEB-E2p53m was created byreplacing the PstI-EcORI region of pWEB-E2, encoding the C-terminalregion of E2, with the corresponding region of pKK-E2p53m encoding themutated C-terminal region of E2 (FIG. 2). The plasmid pVP22 (Invitrogen)encodes the Herpes Simplex Virus-1 VP22 protein. DNA sequences encodingthe E2 and E2p53m proteins were cloned into pVP22 in frame with the VP22coding sequence. All constructs were sequenced to check for the presenceof any unwanted mutations.

b. Protein Purification

The E2Ct and E2p53mCt proteins were expressed in bacteria and purifiedexactly as described previously (Webster K., et al. (2000) J. Biol.Chem. 275, 87-94).

c. Protein-Protein Interactions

In vitro transcription and translation of p53 cloned in pBluescript-p53was carried out using a TNT kit (Promega) according to themanufacturer's instructions. The HPV 16 E2Ct and HPV 16 E2p53mCtproteins were immobilized on PVDF membranes by slot blotting. Afterstaining with 0.1% w/v Ponceau S (Sigma) to conform immobilization themembranes were washed three times in Tris-buffered saline, 0.02% v/vTween 20, 10% w/v dried skimmed milk powder (20 minute washes at 22°C.). The membranes were then incubated with 25 μl of ³⁵S-labelled p53 in10 ml of Tris-buffered saline, 0.02% v/v Tween 20, 10% w/v dried skimmedmilk powder (90 minutes at 22° C.). The membranes were then dipped inmethanol before being left to dry on Whatman paper (22° C. for 15minutes). Bound labelled p53 was visualized using a PhosphorImager.

The affinity of E2 derivatives in accordance with the invention for p53may be quantitatively assesed by surface plasma resonance. Moreparticularly, a GST-p53 fusion protein is captured on a BIACORE SensorChip CM5 flow cell surface that has previously been coated with GSTantibodies using an Amine Coupling Kit (BIACORE). Purified E2 proteinand E2 mutants can then be applied to the surface and their bindingassayed using surface plasmon resonance (Buckle M., et al (1996) Proc.Nat. Acad. Sci. (USA) 93, 889-894).

d. Cell Culture and Transfections

HeLa (HPV18 transformed cervical carcinoma cells), SiHa, CaSki, MEI80,NIH3T3, Saos-2, and MCF-7 cells were maintained in Dulbecco's ModifiedEagle's Medium (DMEM) supplemented with 10% foetal bovine serum (FBS)and penicillin (10⁵ units L⁻¹) and streptomycin (100 mg L⁻¹). 866 cellswere maintained in DMEM supplemented with 5% FBS insulin (5 μg μl⁻¹),hydrocortisone (0.01 μg ml⁻¹), penicillin (10⁵ units L⁻¹) andstreptomycin (100 mg L⁻¹). 808F and 873F cells were maintained in DMEMsupplemented with 5% FBS, insulin (5 μg ml⁻¹), epidermal growth factor(EGF) (0.01 μg ml⁻¹) cholera toxin (0.01 μg ml⁻¹), hydrocortisone (0.4μg ml⁻¹), penicillin (10⁵ units L⁻¹) and streptomycin (100 mg L⁻¹). W12cells were maintained in DMEM, 10% FBS, cholera toxin (0.01 nM),hydrocortisone (0.4 μg ml⁻¹), EGF (0.01 μg ml⁻¹), penicillin (10⁵ unitsL⁻¹) and streptomycin (100 mg L⁻¹). W12 cells required 3T3 cell feedersupport. All cells were maintained in a humidified atmosphere at 37° C.and 5% CO₂.

HeLa cells and ME180 cells are HPV18 transformed cervical carcinomacells; SiHa and CaSki cells are HPV16 transformed cervical carcinomacells, Nih3T3, Sao52, and ncF-7 cells are non-HPV transformed celllines.

For microscopy: twenty four hours prior to transfection the cells wereseeded at a density of 3×10⁵ cells per well onto coverslips in six-wellplates and incubated overnight. The liposome based transfectionreagents, Tfx-20 for 866, Saos-2, and CaSki cells, and Tfx-50 for NIH3T3and SiHa (Promega) were used at a ratio of 3:1 liposome: DNA in 1 ml ofserum-free media per transfection. The remaining cell lines weretransfected using Fugene 6 (Roche). Following transfection andincubation for 30 hours, the cells were washed with phosphate-bufferedsaline (PBS) and fixed using 4% paraformaldehyde for 30 minutes in thedark. After a further wash with PBS, the cells were stained withbisbenzimide (Hoechst no. 33258, Sigma) for 30 minutes in the dark.Finally, the cells were washed with PBS and inverted onto microscopeslides with 15 μl of MOWIOL.

For flow cytometry: HeLa cells (2.3×10⁶) were seeded in 75 cm³ flasksand incubated for twenty four hours prior to transient transfectionusing Fugene 6 (Roche).

e. Microscopy and Imaging

Fluorescence microscopy was carried out using a Leica DM IRBE invertedepi-fluorescent microscope with FITC and DAPI filter sets and a 20×airobjective (Leica).

f. Flow Cytometry

Twenty four hours post-transfection HeLa cells were trypsinized andharvested by centrifugation. Floating (dead) cells were harvested fromthe media. The trypsinized and floating cells were pooled and thenwashed twice with PBS before being resuspended in 1 ml of ice-coldmethanol and incubated at −20° C. for 5 minutes. After centrifugation(10 minutes/2500 rpm in a bench-top centrifuge), the cells wereresuspended in 3 ml of PBS containing 50 μg ml⁻¹ propidium iodide(Sigma) and incubated at 4° C. for 30 minutes. After re-centrifugationthe cells were resuspended in 500 μl of PBS and kept in the dark untilanalysis by flow cytometry (Becton Dickinson FACScalibur).

2. Results.

Blocking the Interaction of E2 and p53

p53 binds to the C-terminal DNA binding domain of the E2 protein(Massimi P., et al. (1999) Oncogene 18, 7748-7754). The structure ofthis domain of E2 bound to DNA has been determined by X-raycrystallography (Hegde R. S. & Androphy E. J. (1998) J. Mol. Biol. 284,1479-1489). A molecular model of the E2-p53 interaction was made usingthe co-crystal structure of p53 and the p53-binding protein 53BP2 as aguide (Gorina S, & Pavletich NP. (1996) Science 274, 1001-1005). Themodelling identified amino acids in the E2 protein (including Asp338,Trp341, and Asp344) that can be superimposed on amino acids present in53BP2 and are important in the p53-53BP2 interaction (FIG. 3). Asp338,Trp341, and Asp344 in E2Ct were mutated to alanine using site-directedmutagenesis. The replacement of an amino acid residue with alanine isconsidered to be a neutral solution. The p53 binding ability of an E2derivative in accordance with the invention may be further reduced bythe introduction of an oppositely-charged residue which actively repelsthe p53 molecule. The ability of the mutated E2p53mCt protein to bindp53 in vitro was tested as described in section lc (FIG. 4). Althoughlabelled p53 binds to the wild-type E2Ct protein, labelled p53 bindsweakly, if at all, to the E2p53mCt protein.

E2p53m Induces Apoptosis in HPV-Transformed Cells but not in non-HPV-Transformed Cells.

The ability of E2-p53m to induce apoptosis was investigated byintroducing the Asp338, Trp341, and Asp344 mutations into the fulllength E2 protein and transiently transfecting the construct into avariety of HPV-transformed and non-HPV transformed cell lines growing oncoverslips (FIG. 5) (as described in Webster et al 2000 infra). Thirtyhours post-transfection the cells were fixed and their DNA was stainedwith bisbenzimide (Hoechst stain). The transfected cells were identifiedon the basis of their green fluorescence upon excitation through afluorescein isothiocyanate filter set. These cells were examined forchromatin condensation and membrane blebbing, two characteristicfeatures of apoptosis, using Hoechst stain and GFP, respectively(Webster K., et al. (2000) J. Biol. Chem. 275, 87-94). One hundredtransfected cells were counted on each coverslip and the experiment wasrepeated three times. All of the cell lines show a background level ofapoptosis of between 3 and 12%; cells transfected with the empty pWEBvector (FIG. 5, column 3). The wild-type E2 protein induces asignificant increase in the level of apoptosis in all of the cell linestested (FIG. 5, column 4). However, the E2p53m protein only induces asignificant increase in the level of apoptosis in the HPV-transformedcells (FIG. 5, column 5). Importantly, both E2 and E2p53m induceapoptosis in W12 cells, a cell line that contains episomal HPV DNA.

VP22 can be Used to Deliver E2 to Target Cells.

To determine whether the Herpes Simplex Virus-1 VP22 protein could beused to deliver E2 and E2 mutants to target cells, DNA sequencesencoding the E2 protein were cloned in frame into a VP22 expressionvector (Invitrogen). Microscopy underestimates the number of apoptoticcells since dead cells detach from the substrate. In contrast, flowcytometry examines all of the cells, including the floating dead cells,and thus gives a better estimate of the number of apoptotic cells. Toexamine the effects of the VP22-E2 expression plasmid on cell survival,we transiently transfected the plasmid into HeLa cells and twenty fourhours post transfection examined the entire population of cells usingflow cytometry (FIG. 7). HeLa cells transfected with the VP22 vectorshow around 10% apoptotic cells (FIG. 7A). In contrast, HeLa cellstransfected with the VP22-E2 expression vector show around 35% apoptoticcells (FIG. 7B). These data show that VP22-E2 fusion proteins arecapable of inducing apoptosis in these cells. To determine whetherVP22-E2 can move from cell to cell and induce apoptosis in the recipient(bystander) cells, we transiently transfected COS-7 cells with theVP22-E2 expression vector described above. The HPV 16 E2 protein doesnot induce apoptosis in COS-7 cells (Webster K., et al. (2000) J. Biol.Chem. 275, 87-94). COS-7 cells transiently transfected with the VP22-E2expression or with a plasmid that expresses VP22 alone were cultured for30 hours. Media from the transfected COS-7 cells was then removed andadded to cultures of HeLa cells. After 24 hours, the HeLa cellpopulations exposed to these conditioned media were examined by flowcytometry. Media from COS-7 cells expressing VP22 alone brings about asmall increase in the percentage of apoptotic HeLa cells; from around8-10% in the untreated population to around 15-20% in the treated cells(FIG. 8A). In contrast, media from COS-7 cells expressing VP22-E2 bringsabout a dramatic increase in the number of apoptotic cells; from around8-10% in the untreated cells to greater than 60% in the treated cells(FIG. 8B). These data suggest that VP22-E2 fusion proteins produced inCOS-7 cells can enter the media from where they are capable of enteringand inducing apoptosis in HeLa cells. Thus cell-cell contact is notrequired for the movement of VP22-E2 into non-producing cells.

Taken together these data suggest that the E2-p53 interaction isnecessary for E2-induced cell death in non-HPV transformed cells. Sincethe E2 protein can induce apoptosis in HPV-transformed cells by alteringthe expression of E6 and E7, mutations that block the interaction of E2with p53 result in an E2 mutant that only induces apoptosis inHPV-transformed cells. These data also show that VP22 can be used todeliver E2 proteins to target cells and that VP22-E2 fusion proteinsproduced in one cell are able to kill bystander cells.

1. A method of killing cells that contain HPV DNA, comprising contactingthe cells with a p53 binding-defective PV E2-derived polypeptide.
 2. Amethod according to claim 1 in which the cells are PV-transformed or PVinfected.
 3. A method according to claim 1 or 2 in which the HPV DNA isintegrated into the cell genome
 4. A method according to claim 1 or 2 inwhich the HPV DNA is present in the cells as episomes.
 5. A methodaccording to claim 1 or 2 in which the HPV DNA is present in the cell asboth episomes and genome integrated DNA.
 6. A method of inducingapoptosis in PV-transformed cells and/or PV-infected cells comprisingcontacting the cells with a p53 binding-defective PV E2-derivedpolypeptide.
 7. A method of killing PV-transformed cells and/orPV-infected cells comprising contacting the cells with a p53binding-defective DNA sequence encoding a p53 binding-defective PVE2-derived polypeptide.
 8. A method of killing PV-transformed cells andPV-infected cells comprising contacting the cells with a p53binding-defective PV E2-derived polypeptide.
 9. A method of killingPV-transformed cells and PV-infected cells comprising contacting thecells with a nucleotide sequence encoding a p53-binding defective PVE2-derived polypeptide.
 10. A method according to claim 9 in which thenucleotide sequence encodes a p53-binding defective PV E2-derivedpolypeptide fused to VP22.
 11. A method according to claim 10 in whichthe p53-binding defective E2 derived polypeptide is delivered as ahistidine-tagged VP22 fusion protein.
 12. A method according to claim 8in which the nucleotide encodes a PV E2-derived polypeptide that isunable to bind p53 fused to histidine-tagged VP22.
 13. A methodaccording to any preceding claim in which more than 50% of the cells arekilled.
 14. A method according to claim 13 in which more than 80% of thecells are killed.
 15. A method according to claim 14 in which more than90% of the cells are killed.
 16. A method according to any precedingclaim in which the cells in which apoptosis is induced are tumourigenic.17. A method according to claim 16 in which the cells are cancerous andin which the cancer is selected from cervical cancer, cancer of thevulva, oral cancer or cancer of the oesophagus.
 18. A method accordingto any preceding claim in which the cells are mammalian.
 19. A methodaccording to claim 18 in which the cells are human cells.
 20. A methodaccording to claim 18 or 19 in which the cells are in a mammaliansubject.
 21. A method according to any preceding claims and in which thep53-binding defective E2 derived polypeptide is able to bind p53 butunable to induce p53 dependent apoptosis.
 22. The use of a p53-bindingdefective PV E2-derived polypeptide in a pharmaceutical composition forthe treatment or amelioration of cervical cancer.
 23. The use of a p53binding defective E2-derived polypeptide in a pharmaceutical compositionfor the treatment or amelioration of genital warts.
 24. The use of a DNAsequence encoding a p53-binding defective E2-derived polypeptide in apharmaceutical composition for the treatment or amelioration of cervicalcancer.
 25. The use of a DNA sequence encoding a p53-binding defectiveE2-derived polypeptide in a pharmaceutical composition for the treatmentor amelioration of genital warts.
 26. A p53-binding defective E2-derivedpolypeptide
 27. A p53 binding-defective E2-derived polypeptide accordingto claim 25 in which the polypeptide is mutated at at least one ofpositions Asp338, Glu340, Trp341, Glu342, Arg343, Asp344 and Glu345 of anative HPV 16 protein amino acid sequence.
 28. A p53-binding defectiveE2-derived polypeptide according to claim 25 or 26 which is unable tobind p53.
 29. A p53-binding defective polypeptide which is derived fromHPV type 16, 18, 33, 31, 6, 11, 2, 4, 1, or 7proteins.
 30. A polypeptideaccording to claim 25, 26,27 or 28 in which the PV is an animal PV. 31.A polypeptide according to claim 29 in which the PV is a mammalian PV.32. A polypeptide according to any one of claims 25 to 30 which has thesame or similar length as a native E2 protein.
 33. A polypeptideaccording to any one of claims 25 to 30 which is substantially shorteror longer than a native E2 protein.
 34. A polypeptide having the aminoacid sequence shown in FIG.
 2. 35. A fusion protein comprising a p53binding-defective PV E2-derived polypeptide and a VP22 fusion sequence.36. A polynucleotide encoding a p53-binding defective E2-derivedpolypeptides according to any one of claim 25 to
 33. 37. Apolynucleotide having the nucleotide sequence of FIG.
 2. 38. Anucleotide encoding an HPV 16 E2 derived polypeptide mutated atpositions Asp338, Glu340, Trp341, Glu342, Arg343, and Asp344 and Glu345of the native sequence or any other positions that are important for theE2-p53 interaction.